Hydroxyurea (HU) has been shown to increase the proportion of fetal hemoglobin (Hb F) in most sickle cell patients. A low dosage regimen of HU increased total hemoglobin (Hb) levels in some thalassemia intermedia patients by preferentially increasing b-globin biosynthesis. We examined the effect of dosage, duration of exposure, and developmental stage at which HU was given on various hematological parameters and signaling pathways. Erythroid cells in two-phase liquid culture were exposed to HU (5 to 100 mM) either as a pulse for 3 days or continuously for 12 days. Low doses of HU (from 0 to 25 mM) increased Hb levels by up to 2.7-fold, and a high dose of HU (100 mM) increased Hb levels when added at days 3-6 of phase II. No significant changes in Hb levels were observed in response to HU during the late stage of phase II culture (>=9-12 days), although there was a significant dose-dependent increase in Hb F levels. HU exposure during days 0-3 of phase II culture increased the number of erythroid colonies to a maximum of 5-fold at 5 mM HU. GATA-1 mRNA was down-regulated at a high dose of HU and GATA-2 was dose-dependently up-regulated over a lower dosage range. Treatment with 100 mM HU dramatically upregulated the death receptor DR-5, caspase 3, and various other genes related to cell cycle control and apoptosis, as determined by cDNA microarray analysis. In contrast, 10 mM HU modestly up-regulated mRNA levels of the early growth response gene (egr-1). Our results suggest that HU exerts concentration-dependent effects on Hb F production and erythropoiesis and that these two effects are mediated by distinct molecular mechanisms. We are currently focusing further clarifying the mechanism of low dose HU on erythropoiesis by studying the Egr-1 signal pathways.